Evaluation of serum vascular endothelial growth factor and endostatin in systemic sclerosis patients – correlation with lung and cardio-vascular system involvement

Correspondence: Bożena Dziankowska-Bartkowiak, MD PhD, Department of Dermatology, Medical University, Krzemieniecka 5, 94-017 Lodz, Poland, tel. +48 42 686 79 81, fax +48 42 688 45 65, e-mail: anuciazalewska@hotmail.com


Introduction
Scleroderma (Sclerodermia Systemica, Systemic Sclerosis, SSc) is a multisystem connective tissue disease with small and larger vessel involvement, which may trigger autoimmunological processes and antinuclear antibodies (ANA) production. Scleroderma is characterized by excessive collagen and other extracellular matrix components production both in the skin and internal organs [1]. Those processes can lead to severe disability or even death [2].
Literature data point out at the importance of angiogenesis regulatory factors in pathogenesis of systemic sclerosis [3, 4]. Angiogenesis i. e. development of new blood vessels is a crucial process in both physiology including embryogenesis, menstrual cycle, decidua formation, hair growth cycle, wound healing and pathology including neoplasms and metastases development, rheumatoid arthritis, diabetic retinopathy, psoriasis vulgaris, endometriosis and connective tissue diseases [5, 6].
Experimental data demonstrated that angiogenesis could be influenced by serum of SSc patients. This process depends on the stage and type of the disease and the balance between pro- and antiangiogenic factors [7].
The following substances are considered to stimulate angiogenesis: basic fibroblast growth factor (bFGF) [8, 9], interleukin 18 (IL-18) [10] and vascular endothelial growth factor (VEGF). VEGF is an angiogenic and mitogenic glicoprotein which increases blood vessel permeability via two selective endothelial cells surface receptors [6]. VEGF after receptor binding, stimulate endothelial cell proliferation, plasminogen activation, induces a and b integrin subunits expression and leads to changes in collagenase activation [6]. Literature data showed that this cytokine could influence proliferation, survival as well as migration of endothelial cells [11, 12]. Changes in endothelial cells in the course of systemic sclerosis, are one of key factors in the development of progressive fibrosis of the skin and internal organisms. It was demonstrated that destruction of some endothelial cells can further stimulate destruction of the other cells. Subsequently, production of different cytokines and adhesion molecules, which activate fibroblasts situated in the vicinity of blood vessels is observed [13].
VEGF plays an important role in many diseases characterized by tissue hypoxia where its expression in greatly enhanced [14-16]. It was demonstrated that angiogenesis observed in neoplastic processes many be stimulated by VEGF and thus responsible for disease progression [17, 18]. Favorable effect of VEGF was found only in a very few cases of peripheral blood vessel diseases and ischaemic heart disease [19].
It was demonstrated that VEGF serum levels correlated with disease activity in both rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) [20].
However, knowledge on the role of VEGF in SSc is considered to be rather inconsistent. On the one hand, VEGF can exert a favorable effect by prevention of digital phalanges ulcer development, but on the other hand it can be responsible for disease severity and progression [21, 22].
Blood vessel atrophy is often observed on capilaroscopy in SSc patients presenting ulcerations. It was observed that inhibition of angiogenesis may be triggered by monocytes and lymphocytes obtained from SSc patients [23, 24]. The following substances exert an inhibitory effect on angiogenesis: angiostatin, endostatin, some interleukins (IL-1, IL-4, IL-10, IL-12), interferons, tissue inhibitors of metalloproteinase 1, 2 and 3 (TIMP-1, -2, -3) as well as angiopoietin 2 and thrombospondin 1 and 2 [25].
Endostatin is a non-collagenic domain of collagen XVIII, of 20 kDa mollecular weight [26]. Considerable amounts of this protein are found in the blood vessels of the lungs and the skin, which are the organs most frequently involved in SSc patients. Collagen XVIII is produced by fibroblasts and hepatocytes and its proteolytic fragment – endostatin is easily found in the circulation after being processed by catepsin L from perivascular collagen [27]. This protein can be found in the blood, urine and basement membranes of many internal organs [28]. Role of endostatin has not been fully elucidated. However, it was demonstrated that endostatin exert a selective inhibitory effect on endothelial cell proliferation stimulated by bFGF [29]. It also decreases activation of proenzymatic metalloproteinase 2 and catalytic properties of membrane metaloproteinases type 1 and 2 [30]. Endostatin can considerably reduce endothelial cell migration to newly developed basement membrane stimulated by VEGF [30]. Endostatin was quite well investigated in neoplastic diseases [25]. Some literature data point at favourable effect of endostatin administration, as a recombinant protein, to the patients with solid tumors [31]. It was observed that incubation of endothelial cells with endostatin leads to cell apoptosis [32, 33].
Studies on the role of endostatin in connective tissue diseases did not find and correlation between this protein serum levels and activity of the disease. Increased endostatin levels were observed in SSc patients in comparison to the control group [4]. Disturbances in VEGF and endostatin production were also observed in patients with rheumatoid arthritis [34].
The above-described data on the role of both VEGF and endostatin, which inevitably exert some role on angiogenesis in scleroderma, prompted us to evaluate both proteins levels in serum of 33 systemic sclerosis patients. Detailed statistical analysis was performed to describe any correlations between protein levels and internal organ changes in the examined group of patients.
Material and methods
Our study involved 33 SSc patients, 28 women and 5 men, age range 16-69 years. In our group 19 patients presented limited SSc – lSSc (mean age 52.7±12.8 years) and 15 diffuse form – dSSc (mean 45.5±12.6 years). All the patients were diagnosed according to ARA criteria [35]. Because of the rapid disease progression, all the dSSc patients were on immunosupressive regimen (50 mg cyclophosphamide daily and 20 mg prednison daily). The patients with lSSc were never put on these drugs. All patients also received channel blockers or pentoxifilline and vitamin E in comparable doses.
Duration of Raynaud’s phenomenon and skin sclerosis were evaluated in all the patients. The degree of sclerosis of the skin was assessed using the Total Skin Score (TSS) according to Kahaleh et al. (0-66 points) [36].
Basic laboratory tests, including erythrocyte sedimentation rate (ESR), full blood count, urine tests, urea and creatinine levels were performed in all the patients. The following additional examinations were also performed in our group of patients: oesophagal scyntigraphy, 24-hour ECG monitoring, Doppler echocardiography, chest X-ray, X-ray of feet and hand bone, lung spirometry. The letter was evaluated in percentages of the required value: Forced Vital Capacity (FVC), Forced Expiratoy Volumen in 1 sec (FEV₁) and the ratio FEV₁/FVC. Restrictive disturbances of pulmonary changes were diagnosed when FVC was below 80% of the desired value, while the value of the ratio FEV₁/FVC was elevated or within the normal range. Obstructing lesions were diagnosed when the value of FEV₁ was reduced, and mixed lesions when the reduction in the predicted values of both FVC and FEV₁ were similar [37].
Antinuclear antibodies serum levels (ANA) were identified by the indirect immunofluorescence method (IIF), using HEp-2 cells as a substrate (Sigma Diagnostics). The precise identification of antibodies was performed by the double immunodiffusion method (DID) in agar gelatin according to Outchterlony [38].
Blood was collected in the morning into the pyrogen-free tubes and stored in -20^oC until further evaluated. Serum levels of VEGF and endostatin were evaluated by immunoenzymatic method (ELISA). Commercially available kits were employed (Quantikine R&D Systems Inc, USA). Concentrations were calculated using a standard curve generated with specific standards provided by the manufacturer. The minimum detecteble concentration of VEGF was less than 9.0 pg/ml and endostatin 1.953 ng/ml.
VEGF levels were evaluated in 33 patients (14 with dSSc and 19 with lSSc) and endostatin in 30 patients (14 with dSSc and 16 with lSSc).
The control group consisted of 20 healthy persons (15 women, 5 man; aged 25-64 years, mean age 46.3±13.2 years).
All the patients and individuals from the control group gave their informed consent to participate in our study according to the requirements of the Bioethic Committee of Medical University of Lodz.
Statistical analysis
The obtained results were expressed as mean, maximum, minimum, and median values together with standard deviation. Numerical variables distribution was assessed by Shapiro-Wilk test, Mann-Whitney, Cochran-Cox and two independent sample tests were employed for comparison of mean values. Correlation between numerical values was evaluated by Spearman rang correlation coefficient – σ. A p value less than 0.05 was considered to be statistically significant.
Results
Characteristics of the patients are presented in Table 1. Table 2 demonstrated abnormalities and changes in internal organs found in the examined group of patients.
The mean value of VEGF in systemic sclerosis patients was 194.0±196.8 pg/ml and in control group 271.2±201.0 pg/ml, in lSSc patients was 219.5±228.4 pg/ml and in dSSc patients 159.6±144.6 pg/ml (Table 3). The differences were not statistically significant (p>0.05).
The mean value of endostatin in systemic sclerosis patients was 113.6±60.7 ng/ml and in the control group 73.6±25.9 ng/ml (Table 3). The difference was statistically significant (p=0.003). Statistically significant difference was also found between endostatin serum levels in the control group and the patients with and dSSc group (p=0.007). The differences in the mean value in both subgroups were not statistically significant (Table 3).
We have not found any statistically significant correlations between VEGF and endostatin serum levels and Raynaud’s phenomenon duration, disease duration, skin lesion severity and number of internal organ involved (Table 4).
Statistical analysis showed that higher VEGF levels were found in patients with changes on chest X-ray examinations (systemic sclerosis – p=0.02; limited SSc – p=0.002), whereas higher endostatin levels were more often observed in patients with disturbances in cardiovascular system (systemic sclerosis – p=0.01; lSSc p=0.006) and disturbed lung function (lSSc – p=0.03) (Table 5).
Discussion
The process of angiogenesis has been much wider studied in neoplastic processes than in connective tissue diseases [39]. However, there is some data on angiogenesis disturbances in the course of autoimmunological processes [40, 41].
Ordinary tissue hypoxia can lead to new blood vessel formation [19]. In scleroderma, on histopathology one can observe perivascular inflammatory infiltrate, diminishing the number of blood vessels and narrowing of their lumen together with excessive deposition of extracellular matrix components [21]. However, despite decreased blood flow and reduced partial oxygen pressure in scleroderma, there is no evidence of proper new blood vessel formation in the skin [22].
It was suggested that VEGF levels may reflect disease activity and that there is some correlation between its serum concentration and lung fibrosis [42]. Viac et al., however, did not observe such correlation [43]. The majority of research groups report that serum VEGF levels in SSc patients are increased in comparison to the control group [3]. In the examined group of patients mean VEGF levels were demonstrated to be lower when comparing to the control group. However, some patients presented higher levels than the control group. Lower VEGF levels were observed in dSSs patients than lSSc ones, which is not in agreement with Distler et al. [3]. Those authors suggested some correlation between higher VEGF levels and disease activity together with disturbed angiogenesis, which may be a proof of biological blockage development of pro-angiogenic activity of VEGF in the systemic sclerosis patients. The role of VEGF receptors in the course of angiogenesis has not been elucidated in our patients. It is widely accepted that many different factors such as hypoxia, interleukin 1, transforming growth factor beta and platelet-derived growth factor influence VEGF activity. In lSSc group we observed that the lower serum VEGF level, the more severe skin fibrosis, whereas in dSSc group the higher VEGF level the more severe skin fibrosis. It is difficult to explain the above differences. Maybe they result from different VEGF levels observed in patients with different duration of Raynaud’s phenomenon and duration of the disease itself. In dSSc group the shorter Raynaud’s phenomenon and disease duration the higher VEGF levels were found in contrast to the lSSc group. Our results seem to support the hypothesis of dual function of VEGF in pathogenesis of systemic sclerosis. On the one hand, VEGF can exert a favorable effect on blood vessels on the other hand, it can precipitate skin fibrosis development [3]. In RA patients median serum levels of VEGF were comparable with those found for SSc patients in Distler et al study [3]. Some authors observed that blockade of VEGF reduced the disease severity in murine collagen-induced arthritis [44, 45].
Statistically significant difference between VEGF levels and internal organ changes was found in all the examined lSSc subgroup. Higher VEGF levels correlated with changes observed on chest X-ray which is in agreement with the results of Kikuchi et al. [42]. This group suggested that VEGF, via endothelial cells influences remodeling of blood vessels in the lungs, which in turn leads to lung fibrosis.
Proper angiogenesis depends on the balance between pro- and anti-angiogenic factors. So, there are research works aiming at explanation of these process disturbances in the course of scleroderma and other connective tissue diseases. Correlation between endostatin levels and other factors is still not fully elucidated. VEGF exert its action on the release of different collagenases and other substances, which cleave endostatin from collagen XVIII, from endothelial cells [46, 47]. Increased endostatin levels were found in rheumatoid arthritis [48] and systemic lupus erythematosus [20]. Hebbar et al. observed some correlation between serum endostatin levels and cutaneous ulcers or scars developed in SSc patients [4]. Other authors however, have only found increased serum endostatin levels in a few patients without any correlation with clinical parameters [3]. Differences in the observed results are not clarified.
Our results demonstrated statistically significantly higher serum endostatin levels in SSc patients than in the control group. There were some differences in endostatin levels between limited and diffuse SSc patients, however they did not reach statistical significance. There was a strong but statistically insignificant, correlation between disease duration and skin fibrosis severity and increased serum endostatin levels. Lack of statistical significance could be the result of a relatively small number of the examined patients. Hebbar et al [4] and Hunzelmann et al [49] observed that in systemic sclerosis patients endostatin serum levels were increased and correlated with more extensive skin involvement.
In the course of scleroderma, collagen XVIII can be detected in the perivascular basement membrane zone of the skin and many internal organs involved [50]. Higher endostatin levels were observed in the examined SSc patients with the gastrointestinal tract, heart, lungs and osteo-articular systems involvement. However, only correlation between endostatin levels and cardiovascular system disturbances and pulmonary dysfunction found on spirometry, have reached statistical significance. It could be assumed that despite not full elucidation of endostatin action, this protein can exert an inhibitory effect on pro-enzymatic metalloproteinase 2. It can also inhibit catalytic action on membrane metalloproteinases type 1 and 2 [3]. In this way, endostatin can favor excessive deposition of extracellular matrix components and fibrosis development in the internal organs [30]. Hebbar et al also found that some patients with pulmonary fibrosis, which was detectable on chest radiograms, had higher endostatin concentrations [4]. These authors suggest that endostatin detected is released by tissue-activated fibroblasts.
Quite interesting is another observation i. e. the lower serum VEGF levels the higher serum endostatin levels.
Our research showed that disturbances in levels of pro- and antiangiogenic factors play an important role in pathogenesis of systemic sclerosis and their serum levels can correlate with lung and cardiovascular system involvement.
References
1. Sollberg S, Krieg T (1996): New aspects in scleroderma research. Int Arch Allergy Immunol 111: 330.
2. Fimiani C (2001): Systemic sclerosis: a woman disease. Mod Asp Immunobiol 1: 233.
3. Distler O, del Rosso A, Giacomelli R, Cipriani P, Conforti ML, Guiducci S, Gay RE, Michel BA, Bruhlmann P, Muller-Ladner U, Gay S, Matucci-Cerinic M (2002): Angiogenic and angiostatic factors in systemic sclerosis: increased levels of vascular endothelial growth factor are a feature of earliest disease stages and are associated with the absence of fingertip ulcers. Arthritis Res 4: R11.
4. Hebbar M, Peyrat JP, Hornez L, Harton PY, Hachula E, Devulder B (2000): Increased concentrations of circulating angiogenesis inhibitor endostatin in patients with systemic sclerosis. Arthritis Rheum 43: 889.
5. Velasco P, Lange-Asschenfeldt B (2002): Dermatological aspects of angiogenesis. Br J Dermatol 147: 841.
6. Josko J, Gwozdz B, Jedrzejowska-Szypulka H, Hendryk S (2000): Vascular endothelial growth factor (VEGF) and its effect on angiogenesis. Med Sci Monit 6: 1047.
7. Majewski S, Skopinska-Rozewska E, Jablonska S, Polakowski I, Pawinska M, Marczak M, Szmurlo A (1985): Modulatory effect of sera from scleroderma patients on lymphocyte-induced angiogenesis. Arthritis Rheum 28: 1133.
8. Dvorak HF, Detmar M, Claffey KP, Nagy JA, van de Water L, Senger DR (1995): Vascular permeability factor/vascular endothelial growth factor: an important mediator of angiogenesis in malignancy and inflammation. Int Arch Allergy Immunol 107: 233.
9. Pepper MS (1997): Manipulating angiogenesis. From basic science to the beside. Artherioscler Thromb Vasc Biol 17: 605.
10. Park CC, Morel JC, Amin MA, Connors MA, Harlow LA, Koch AE (2001): Evidence of IL-18 as a novel angiogenic mediator. J Immunol 167: 1644-1653.
11. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, Declercq C, Pawling J, Moons L, Collen D, Risau W, Nagy A (1996): Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380: 435.
12. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, Powell-Braxton L, Hillan KJ, Moore MW (1996): Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380: 439.
13. Mittag M, Beckheinrich P, Haustein UF (2001): Systemic sclerosis-related Raynaud’s phenomenon: effects of Illoprost infusion therapy on seru cytokine, growth factor and soluble adhesion molecule levels. Acta Derm Venereol 81: 294.
14. Christou H, Yoshida A, Arthur V, Morita T, Kourembanas S (1998): Increase vascular endothelial growth factor production in the lings of rats with hypoxia-induced pulmonary hypertension. Am J Respir Cell Mol Biol 18: 768.
15. Sandner P, Wolf K, Bergmaier U, Gess B, Kurtz A (1997): Induction of VEGF and VEGF receptor gene expression by hypoxia: divergent regulation in vivo and in vitro. Kidney Int 51: 448.
16. Adamis AP, Miller JW, Bernal MT, D’Amico DJ, Folkman J, Yeo TK, Yeo KT (1994): Elevated vascular permeability factor/vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retnopathy. Am J Ophtalmol 118: 445.
17. Kieran MW, Billett AM (2001): Antiangiogenesis therapy. Hem/Oncol Clin North Am 15: 835.
18. Folkman J (1995): Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1: 27.
19. Matucci-Cerinic M, Generini S, Pignone A (1997): New approaches to Raynaud’s phenomenon. Curr Opin Rheumatol 544: 9.
20. Robak E, Woźniacka A, Sysa-Jedrzejowska A, Stepien H, Robak T (2002): Circulating angiogenesis inhibitor endostatin and postive endothelial growth regulators in patients with systemic lupus erythematosus. Lupus 11: 348.
21. Furst DE, Clements PJ (1996): Pathogenesis, fusion. In Systemic sclerosis (Edited by: Furst DE, Clements PJ). Baltimore, MD: Williams & Wilkins: 275.
22. LeRoy EC (1996): Systemic sclerosis. A vascular perspective. Rheum Dis Clin North Am 22: 675.
23. Koch AE, Litvak MA, Burrows JC, Polverini PJ (1992): Decreased monocyte-mediated angiogenesis in scleroderma. Clin Immunol Immunopathol 64: 153.
24. Kaminski MJ, Majewski S, Jablonska S, Pawińska M (1984): Lowered angiogenic capability of peripheral blood lymphocytes in progressive systemic sclerosis (scleroderma). J Invest Dermatol 82: 239.
25. Szary J, Szala S (2002): Endostatyna w terapii nowotworów. (in Polish) J Oncol 52: 159.
26. Zatterstrom UK, Felbor U, Fukai N, Olsen BR (2000): Collagen XVIII/endostatin structure and functional role in angiogenesis. Cell Structure and Function 25: 97.
27. Felbor U, Dreier L, Bryant RA, Ploegh HL, Olsen BR, Mothes W (2000): Secreted cathepsin L generates endostatin from collagen XVIII. EMBO J 19: 1187.
28. Sasaki T, Fukai N, Mann K, Gohring W, Olsen BR, Timpl R (1998): Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin. EMBO J 17: 4249.
29. O’Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J (1997): Endostatin an endogenous inhibitor of angiogenesis and tumor growth. Cell 88: 277.
30. Kim YM, Jang JW, Lee OH, et al. (2000): Endostatin inhibits endothelial and tumor cellular invasion by blocking the activation and catalytic activity of matrix metalloproteinase 2. Cancer Res 60: 5410.
31. Carmelit P, Jain RK (2000): Angiogenesis in cancer and other diseases. Nature 407: 249.
32. Dixelius J, Larsson H, Sasaki T, Holmqvist K, Lu L, Engstrom A, Timpl R, Welsh M, Claesson-Welsh L (2000): Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis. Blood 95: 3403.
33. Dhanabal M, Ramchandran R, Waterman MJ, Lu H, Knebelmann B, Segal M, Sukhatme VP (1999): Endostatin induces endothelial cell apoptosis. J Biol Chem 274: 11721.
34. Nagashima M, Asano G, Yoshino S (2000): Imbalance in production between vascular endothelial growth factor and endostatin in patients with rheumatoid arthritis. J Rheumatol 27: 2339.
35. Subcommittee for scleroderma Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. 1980. Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 23: 581.
36. Kahaleh MB, Sultany GL, Smith EA, Huffstutter JE, Loadholt CB, LeRoy EC (1986): A modified scleroderma skin score method. Clin Exp Rheumatol 4: 367.
37. Martinez FJ (2000): Badania czynnościowe układu oddechowego. In: Urban&Partner (eds). Choroby płuc. Diagnostyka I terapia. Khan MG, Lynch JP III, 1st edition (Polish): 103.
38. Rofstad EK, Danielsen T (1998): Hypoxia-induced angiogenesis and vascular endothelial growth factor secretion in human melanoma. Br J Cancer 77: 987.
39. Robak E, Wozniacka A, Sysa-Jedrzejowska A, Stepien H, Robak T (2001): Serum levels of angiogenic cytokines in systemic lupus erythematosus and their correlation with disease activity. Eur Cytokine Netw 12: 445.
40. Kikuchi K, Kubo M, Kadano T, Yazawa N, Ihn H, Tamaki K (1998): Serum concentrations of vascular endothelial growth factor in collagen diseases. Br J Dermatol 139: 1049.
41. Viac J, Schmitt D, Claudy A (2000): Plasma Vascular Endothelial growth Factor levels in scleroderma are not correlated with disease activity. Acta Derm Venereol 80: 383.
42. Ballara S, Taylor PC, Reusch P, Marme D, Feldmann M, Maini RN, Paleolog EM (2001): Raised serum vascular endothelial growth factor levels are associated with destructive change in inflammatory arthritis. Arthritis Rheum 44: 2055.
43. Miotla J, Maciewicz R, Kendrew J, Feldmann M, Paleolog E (2000): Treatment with soluble VEGF receptor reduces disease severity in murine collagen-induced arthritis. Lab Invest 80: 1195.
44. Lamoreaux WJ, Fitzgerald ME, Reiner A, Hasty KA, Charles ST (1998): Vascular endothelial growth factor increase release of gelatinase A and decrease release of tissue inhibitor of metalloproteinases by microvascular endothelial cells in vitro. Microvasc Res 55: 29.
45. Zucker S, Mirza H, Conner CE, Lorenz AF, Drews MH, Bahou WF, Jesty J (1998): Vascular endothelial growth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: controversion of prothrombin to thrombin results in progelatinase A activation and cell proliferation. Int J Cancer 75: 780.
46. Nagashima M, Asano G, Yoshino S (2000): Imbalance in production between vascular endothelial growth factor and endostatin in patients with rheumatoid arthritis. J Rheumatol 27: 2339.
47. Hunzelmann N, Risteli J, Risteli L, Sacher C, Vancheeswaran R, Black C, Krieg T (1998): Circulating type I collagen degradation products: a new serum marker for clinical severity in patients with scleroderma? Br J Dermatol 139: 1020.
48. Hafler W, Dong S, Schurer B, Cole GL (1998): Collagen XVIII is a basement membrane heparin sulfate proteoglycan. J Biol Chem 25: 25404.

This work was supported by Personal grant no.
502-11-697 from a Medical University of Lodz.